The present invention pertains generally to viral diagnostics. In particular, the invention relates to an antigen/antibody combination assay for accurately diagnosing hepatitis C virus infection.
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis (NANBH) which is transmitted largely through blood transfusion and sexual contact. The virus is present in 0.4 to 2.0% of blood donors. Chronic hepatitis develops in about 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control of the disease is of medical importance.
HCV was first identified and characterized as a cause of NANBH by Houghten et al. The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, as shown in FIG. 1, several proteins are encoded by the HCV genome. The order and nomenclature of the cleavage products of the HCV polyprotein is as follows: NH2-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed xe2x80x9ccorexe2x80x9d) and two envelope glycoproteins, xe2x80x9cE1xe2x80x9d (also known as E) and xe2x80x9cE2xe2x80x9d (also known as E2/NS1), as well as nonstructural (NS) proteins that contain the viral enzymes. The NS regions are termed NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity. NS2, either alone or in combination with NS3, cleaves the NS2-NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease serves to process the remaining polyprotein. Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease. Subsequent NS3-mediated cleavages of the HCV polyprotein appear to involve recognition of polyprotein cleavage junctions by an NS3 molecule of another polypeptide. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNA polymerase (NS5b).
A number of general and specific polypeptides useful as immunological and diagnostic reagents for HCV, derived from the HCV polyprotein, have been described. See, e.g., Houghton et al., European Publication Nos. 318,216 and 388,232; Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364; Houghton et al., Hepatology (1991) 14:381-388; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D. Y., International Publication No. WO 94/01778. These publications provide an extensive background on HCV generally, as well as on the manufacture and uses of HCV polypeptide immunological reagents. For brevity, therefore, the disclosure of these publications is incorporated herein by reference.
Sensitive, specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products would provide an important advance in medicine. Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfused patients, and HCV has accounted for up to 90% of these cases. Patient care as well as the prevention and transmission of HCV by blood and blood products or by close personal contact require reliable diagnostic and prognostic tools. Accordingly, several assays have been developed for the serodiagnosis of HCV infection. See, e.g., Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364; Choo et al., Br. Med. Bull. (1990) 46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel et al., Lancet (1990) 335:558-560; van der Poel et al., Lancet (1991) 337:317-319; Chien, D. Y., International Publication No. WO 94/01778; Valenzuela et al., International Publication No. WO 97/44469; and Kashiwakuma et al., U.S. Pat. No. 5,871,904.
A significant problem encountered with some serum-based assays is that there is a significant gap between infection and detection of the virus, often exceeding 80 days. This assay gap may create great risk for blood transfusion recipients. To overcome this problem, nucleic acid-based tests (NAT) that detect viral RNA directly, and HCV core antigen tests that assay viral antigen instead of antibody response, have been developed. See, e.g., Kashiwakuma et al., U.S. Pat. No. 5,871,904; Beld et al., Transfusion (2000) 40:575-579.
However, there remains a need for sensitive, accurate diagnostic and prognostic tools in order to provide adequate patient care as well as to prevent transmission of HCV by blood and blood products or by close personal contact.
The present invention is based in part, on the finding that HCV seroconversion antibodies are typically anti-core and anti-NS3 (helicase). Accordingly, the invention provides an HCV core antigen and NS3 antibody combination assay that can detect both HCV antigens and antibodies present in a sample using a single solid matrix.
Accordingly, in one embodiment, the subject invention is directed to an immunoassay solid support comprising at least one HCV anti-core antibody and at least one isolated HCV NS3/4a epitope bound thereto. The antibody and NS3/4a epitope can be any of the herein described molecules. Additionally, the solid support may include any of the multiple epitope fusion antigens described herein, such as the multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F.
In certain embodiments, the solid support comprises at least two HCV anti-core antibodies bound thereto. Moreover, the anti-core antibody may be a monoclonal antibody. Additionally, the NS3/4a epitope may be a conformational epitope, such as a conformational NS3/4a epitope comprising the amino acid sequence depicted in FIGS. 4A-4D.
In another embodiment, the invention is directed to an immunoassay solid support comprising at least two HCV anti-core monoclonal antibodies and at least one HCV NS3/4a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D, bound thereto.
In still a further embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support as described above; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least one anti-core antibody and the NS3/4a epitope, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least one anti-core antibody bound to the solid support; (ii) an antigen that reacts with an HCV antibody from the biological sample reactive with the NS3/4a epitope; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the antigen of (ii); and (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample. The NS3/4a epitope may be a conformational epitope, such as a conformational epitope having the NS3/4a sequence depicted in FIGS. 4A-4D.
In yet another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support with at least two HCV anti-core antibodies bound thereto, as described above; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies and the NS3/4a epitope, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the hSOD amino acid sequence; and (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample. The NS3/4a epitope may be a conformational epitope, such as a conformational epitope having the NS3/4a sequence depicted in FIGS. 4A-4D.
In any of the above embodiments, the anti-core antibody may be directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence, and/or the detectably labeled HCV anti-core antibody may be directed against a C-terminal region of the HCV core antigen, such as amino acids 120-130 of HCV, numbered relative to the HCV1 polyprotein sequence. Moreover, the antigen that reacts with an HCV antibody from the biological sample may be from the NS3 region, such as an epitope from the c33c region of the HCV polyprotein and can be fused with a human superoxide dismutase (hSOD) amino acid sequence. In this embodiment, the second detectably labeled antibody is reactive with the hSOD amino acid sequence.
In another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support including two HCV anti-core monoclonal antibodies and a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies and the NS3/4a conformational epitope, respectively; adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least two anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with said hSOD amino acid sequence; detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.
In certain embodiments, the at least two anti-core antibodies are directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein, and the detectably labeled HCV anti-core antibody is directed against a C-terminal region of the HCV core antigen, such as against amino acids 120-130 of HCV, numbered relative to the HCV1 polyprotein sequence.
In another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support which includes a multiple epitope fusion antigen; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least one anti-core antibody, the NS3/4a epitope, and the multiple epitope fusion antigen; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least one anti-core antibody bound to the solid support; (ii) first and second antigens that react with an HCV antibody from the biological sample reactive with the NS3/4a epitope and the multiple epitope fusion antigen, respectively; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the antigens of (ii); (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.
The anti-core antibody may be directed against an N-terminal region of the HCV core antigen and said first detectably labeled HCV anti-core antibody may be directed against a C-terminal region of the HCV core antigen, as described above. Moreover, the first antigen that reacts with an HCV antibody from the biological sample may comprise an epitope from the c33c region of the HCV polyprotein, and may be fused with an hSOD amino acid sequence. In this context, the second detectably labeled antibody is reactive with the hSOD amino acid sequence. Additionally, the second antigen that reacts with an HCV antibody from the biological sample may comprise an epitope from the c22 region of the HCV polyprotein, such as an epitope comprising amino acids Lys10 to Ser99 of the HCV polyprotein, with a deletion of Arg47 and a substitution of Leu for Trp at position 44, numbered relative to the HCV1 polyprotein sequence. The epitope may be fused with an hSOD amino acid sequence. If so, the second detectably labeled antibody is reactive with the hSOD amino acid sequence. The multiple epitope fusion antigen may comprise the amino acid sequence depicted in FIGS. 7A-7F.
In yet a further embodiment, the invention is directed to a method of detecting HCV infection in a biological sample, said method comprising: (a) providing an immunoassay solid support which comprises two HCV anti-core monoclonal antibodies, an HCV NS3/4a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D, and a multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F, bound thereto; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies, the NS3/4a conformational epitope, and the multiple epitope fusion antigen, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least two anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence and an epitope from the c22 region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein said second detectably labeled antibody is reactive with said hSOD amino acid sequences; (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.
In this embodiment, the at least two anti-core antibodies may be directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein, and the detectably labeled HCV anti-core antibody is directed against a C-terminal region of the HCV core antigen, such as against amino acids 120-130 of HCV, numbered relative to the HCV1 polyprotein sequence. Moreover, the epitope from the c22 region may comprise amino acids Lys10 to Ser99 of the HCV polyprotein, with a deletion of Arg47 and a substitution of Leu for Trp at position 44, numbered relative to the HCV1 polyprotein sequence.
In other embodiments, the invention is directed to immunodiagnostic test kits comprising the immunoassay solid support described above, and instructions for conducting the immunodiagnostic test.
In still further embodiments, the invention is directed to methods of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding at least one HCV anti-core antibody, such as one or two or more, and at least one isolated HCV NS3/4a epitope thereto, and optionally, a multiple epitope fusion antigen thereto. The anti-core antibodies, NS3/4a epitopes and multiple epitope fusion antigens are as described above.
In additional embodiments, the invention is directed to a multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F, or an amino acid sequence with at least 80% sequence identity, such as 90% or more sequence identity, thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. In certain embodiments, the multiple epitope fusion antigen consists of the amino acid sequence depicted in FIGS. 5A-5F.
In further embodiments, the invention is directed to a polynucleotide comprising a coding sequence for the multiple epitope fusion antigen above, a recombinant vectors comprising the polynucleotides, host cells transformed with the recombinant vectors, and methods of producing a recombinant multiple epitope fusion antigen comprising: (a) providing a population of host cells as above; and (b) culturing the population of cells under conditions whereby the multiple epitope fusion antigen encoded by the coding sequence present in the recombinant vector is expressed.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions, and are therefore incorporated by reference in their entirety.